Active-Site Structure Analysis of Recombinant Human Inducible Nitric

David D. Davey, Marc Adler, Damian Arnaiz, Keith Eagen, Shawn Erickson, William Guilford, Margaret Kenrick, Michael M. Morrissey, Mike Ohlmeyer, Gongh...
0 downloads 0 Views 419KB Size
Download PDF
Biochemistry 1996, 35, 9567-9575

9567

Active-Site Structure Analysis of Recombinant Human Inducible Nitric Oxide Synthase Using Imidazole Renee M. Chabin,‡ Ermenegilda McCauley,§ Jimmy R. Calaycay,§ Theresa M. Kelly,‡ Karen L. MacNaul,§ Gloria C. Wolfe,§ Nancy I. Hutchinson,§ Sayyaparaju Madhusudanaraju,§ John A. Schmidt,§ John W. Kozarich,‡ and Kenny K. Wong*,‡ Departments of Biochemistry and Inflammation Research, Merck Research Laboratories, Rahway, New Jersey 07065 ReceiVed February 27, 1996; ReVised Manuscript ReceiVed May 17, 1996X

ABSTRACT:

Nitric oxide synthase catalyzes the pyridine nucleotide-dependent oxidation of L-arginine to nitric oxide and L-citrulline. It is a specialized cytochrome P450 monooxygenase that is sensitive to inhibition by imidazole. Steady-state kinetic studies on recombinant human inducible nitric oxide synthase (rHiNOS) demonstrate that imidazole and 1-phenylimidazole are competitive and reversible inhibitors versus L-arginine. Structure-activity relationship and pH dependence studies on the inhibition suggest that the neutral form of imidazole may be the preferred species and that the only modifications allowed without the loss of inhibition are at the N-1 position of imidazole. Optical spectrophotometric studies of rHiNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting Kd values of 63 ( 2 and 28 ( 3 µM, respectively. These values were in good agreement with the steady-state Ki of 95 ( 10 and 38 ( 4 µM, respectively, and confirms the site of binding is at the sixth axial ligand of the heme. Imidazole (2.2 mM) also perturbed the Kd of L-arginine from 3.03 ( 0.45 to 209 ( 10 µM. The observed increase in the Kd for L-arginine is consistent with imidazole being a competitive inhibitor versus L-arginine. The IC50 values of imidazole and 1-phenylimidazole were lower in the absence of exogenous BH4, and both inhibitors also competitively inhibited the BH4-dependent activation of the enzyme. These data taken together suggest that the L-arginine, dioxygen, and the BH4 binding sites are in close proximity in rH-iNOS. Furthermore, these studies demonstrate the usefulness of imidazole compounds as active site probes for recombinant human iNOS.

Nitric oxide (NO‚)1 is a biologically important free radical that mediates a variety of signaling processes in mammals. Its reactivity with iron may well be the molecular basis for its many regulatory functions. The best known example is the maintenance of vascular tone where NO‚ activates guanylate cyclase via its binding to the heme of the enzyme. The resulting increase of cyclic GMP leads to vasorelaxation. NO‚ at low to moderate (low micromolar) concentrations is beneficial, but at high concentration (hundreds of micromolar) it becomes destructive, binding to and inhibiting a variety of heme- and iron-containing proteins (Granger & Lehninger, * Corresponding author: Department of Biochemistry, Merck Research Labs, RY80-136M Rahway, NJ 07065. Telephone: (908) 5946114. Fax: (908) 594-3695. E-mail: [email protected]. ‡ Department of Biochemistry. § Department of Inflammation Research. X Abstract published in AdVance ACS Abstracts, July 1, 1996. 1 Abbreviations: NO‚, nitric oxide; iNOS, inducible nitric oxide synthase; cNOS, constitutive nitric oxide synthase; rH-iNOS, recombinant human inducible nitric oxide synthase; rH-ecNOS, recombinant human endothelial nitric oxide synthase; rH-ncNOS, recombinant human neuronal nitric oxide synthase; CaM, calmodulin; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; Bis-Tris propane, 1,3-bis[tris(hydroxymethyl)methylamino]propane; DEAE, diethylaminoethyl anion exchange chromatography; DMSO, dimethyl sulfoxide; β-NADPH, β-nicotinamide adenine dinucleotide phosphate, reduced form; BSA, bovine serum albumin; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; 2′, 5′-ADP, 2′, 5′-adenine dinucleotide phosphate; DTT, dithiothreitol; BH4, 6(R)-5,6,7,8-tetrahydrobiopterin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC, highperformance liquid chromatography, V/Karginine, first order rate constant for L-arginine; Varginine, zero order rate constant for L-arginine.

S0006-2960(96)00476-X CCC: $12.00

1982; Nakaki et al., 1990; Lepoivre et al., 1992). The nondiscriminatory inhibition of essential iron containing proteins serves to destroy invading organisms. However, the prolongation of high concentrations of NO‚ inevitably causes the destruction of the host cells (Mulligan et al., 1991). The constitutive endothelial (NOS 3) and neuronal nitric oxide (NOS 1) synthases are responsible for the generation of NO‚ at regulatory levels and their activities are controlled by Ca2+/calmodulin. The isoform responsible for the generation of destructive levels of NO‚ is the inducible nitric oxide synthase (NOS 2) whose activity is independent of Ca2+/calmodulin regulation but contains tightly bound calmodulin (Cho et al., 1992). Generation of excessive levels of NO‚ by iNOS has been implicated in several inflammatory diseases (Moncada & Higgs, 1993; Cash & Klippel, 1994); thus, a selective inhibitor of iNOS could conceivably modulate such diseases. It has been demonstrated that NOS is a highly specialized cytochrome P450 possessing two hemes per homodimer and carries out two successive hydroxylations of L-arginine (White & Marletta, 1992; Stuehr & Ikeda-Saito, 1992). It is unusual that the functional enzyme consists of an intrinsic P450 reductase domain along with the P450 heme domain in a single polypeptide. Also the enzyme requires BH4 for maximal activity and stability (Hevel & Marletta, 1992; Baek et al., 1993; Mayer & Werner, 1995). In the isolated enzyme, the iron of the heme is predominantly (∼80%) in the high spin state (McMillan & Masters, 1993), in contrast to the other cytochrome P450 enzymes which exist predominantly in the low spin state. Imidazole is a classic inhibitor of © 1996 American Chemical Society

9568 Biochemistry, Vol. 35, No. 29, 1996 cytochrome P450 and has been demonstrated to be an inhibitor of the constitutive and inducible NOS isoforms from different species (Wolff et al., 1993; Wolff & Gribin, 1994a,b; Mayer et al., 1994). However, studies with the rat and porcine brain NOS yielded conflicting results with respect to the binding of imidazole to the P450 heme relative to the binding of L-arginine (Wolff & Gribin, 1994a; Wolff et al., 1994; Mayer et al., 1994). The understanding of how inhibitors bind to the heme active site is paramount in the design of selective inhibitors for the human enzyme. Unfortunately, insufficient amounts of the human iNOS have hampered detailed enzymological analysis. The recent availability of milligram quantities of the recombinant human iNOS (rH-iNOS) has allowed us to initiate biochemical and biophysical studies of this relevant target.2 Therefore, we have investigated the inhibition of purified rH-iNOS with imidazole to probe the relationship between the dioxygen, L-arginine, and BH4 binding sites. We report a thorough kinetic and spectrophotometric study of the inhibition of rH-iNOS by imidazole and 1-phenylimidazole. The data suggest that the L-arginine and BH4 binding sites are in close proximity to the dioxygen binding site of the heme. MATERIALS AND METHODS Chemicals. TES, MES, Bis-Tris propane, imidazole, DMSO, L-arginine hydrochloride, L-citrulline, L-ornithine hydrochloride, β-NADPH, BSA, CaCl2, calmodulin, FAD, FMN, and DTT were purchased from Sigma (St. Louis, MO). Citric acid, sodium azide, and potassium phosphate monobasic were purchased from Fisher Scientific (Springfield, NJ). BH4 was obtained from Dr. B. Schircks (Jona, Switzerland). L-[2,3,4,5-3H]Arginine hydrochloride was purchased from Amersham (Arlington Heights, IL). In-Flow high salt capacity Tru-Count scintillation cocktail was purchased from IN/US Systems Inc. (Fairfield, NJ). All imidazole compounds were purchased from either Sigma or Aldrich or were obtained from the Merck chemical collection. Human Recombinant NOS Isoforms. rH-iNOS was purified from Baculovirus infected Sf9 cells containing the cDNA for the enzyme. Cell lysate was obtained by homogenation of cells in lysis buffer (20 mM TES, pH 7.4, 1 mM DTT, 10% glycerol, 50 µM BH4, 50 µM FAD, 50 µM FMN, 25 µg/mL each of antiplatin, aprotinin, leupeptin and chymostatin, 10 µg/ml pepstatin A, 100 µM PMSF, and 50 µM phenanthroline). Preliminary kinetic studies were performed on enzyme purified to greater than 80% purity by a procedure described by Bredt and Synder (1990) employing a 2′,5′ADP affinity column and a gel filtration column. Most of the studies reported were performed on immunoaffinity purified rH-iNOS. This purification procedure employed an immunoaffinity column constructed by cross-linking an antibody against the carboxy-terminal portion of human iNOS to protein-A agarose followed by a DEAE step. Details of the cloning, expression, and purification of rHiNOS will be reported elsewhere.2 The purity of the enzyme was greater than 95% as judged by SDS-PAGE and had a specific activity of 100 nmol/min mg at 37 °C. The constitutive human isoforms were also from Baculovirus infected Sf9 cells containing either the cDNA for the endothelial or the neuronal enzyme. Cell lysate was prepared 2

Calaycay et al., submitted for publication.

Chabin et al. by the same procedure as that of rH-iNOS except 10 mM CHAPS and 100 mM NaCl were included into the lysis buffer for rH-ecNOS. Assay for Nitric Oxide ActiVity. NOS activity was assayed based on a modification of a published procedure (Robertson et al., 1993). Enzyme incubations consisted of 1-5 µg of immunoaffinity purified iNOS in 100 mM TES, pH 7.5, 5 µM FAD, 5 µM FMN, 2 mM CaCl2, 10 µg/mL CaM, 0.5 mM DTT, 0.5 mM NADPH, 0.5 mg/ml BSA, 0.25 µCi of L-[2,3,4,5-3H] arginine, with 10 µM BH4 (unless otherwise indicated), and with 1 µM Arg (unless otherwise indicated). All reactions were incubated at room temperature (20-25 °C) for 30 min and quenched with 200 mM citric acid, pH 2.2, 0.2 mM L-citrulline, 0.2 mM L-ornithine, 0.2 mM L-arginine, and 0.02% NaN3. L-Arginine to L-citrulline conversion was quantitated using a radiometric HPLC assay at room temperature on a Zorbax 300-SCX column (25 cm × 4.6 mm i.d.). A linear gradient of 0-60% 0.1 M KH2PO4 (pH 4.5) for 2 min, followed by a 6 min wash with 0.1 M KH2PO4 (pH 4.5) at a flow rate of 2 mL/min, was used to elute L-[2,3,4,5-3H]Arg and L-[2,3,4,5-3H]Cit. Enzyme activity was linear for 30 min and reported as (% CPM Cit)/ (% CPM Cit + % CPM Arg). The HPLC system consisted of two Shimadzu LC-10AD pumps, a Shimadzu SCL-10A gradient controller, a Shimadzu SIL-10A autosampler, and an IN/US β-RAM radioactivity flow monitor. Data were digitized and analyzed on Shimadzu EZCHROM chromatographic software. Inhibition Studies. The inhibition constants (IC50) of substituted imidazoles were determined by measuring the amount of L-citrulline formed after 30 min in the absence and the presence of increasing concentrations of inhibitor. IC50 values were determined graphically by plotting the inhibitor concentration versus the activity using Sigma Plot (Jandel Scientific). The values were calculated by fitting the data to y ) 1/(1 + x/b), where x is the inhibitor concentration, y is the relative activity, and b is the IC50 value (Segel, 1975). For pH studies, 100 mM TES, pH 7.5, was replaced with 100 mM Bis-tris-propane ranging from pH 6.5 to 9.5. For Ki determinations, time points were taken at 0, 5, 10, 15, and 20 min intervals after initiation of the reactions with enzyme. Initial rates were determined by taking the linear portion of the progress curve at each concentration of L-arginine and a fixed concentration of inhibitor. The data were analyzed and the Ki value obtained from fitting the data to the V ) VA/[K(1 + I/Ki) + A] using the Fortran program of Cleland (Segel, 1975; Cleland, 1979). The cytosolic fraction of murine fibroblast NIH 3T3 cell line expressing rH-iNOS (Tzeng et al., 1995) was used for the examination of the effects of BH4 on the concentrationdependent inhibition of rH-iNOS by imidazole and 1-phenylimidazole. Assay conditions were the same as that described for IC50 determination except that 25 µM L-arginine and 100 nM or 5 µM BH4 were used. Diaphorase ActiVity. The intrinsic diaphorase activity of rH-iNOS was assayed using a modified version of a previous method (Klatt et al., 1992). rH-iNOS was added to a solution that consisted of 100 mM TES (pH 7.5), 500 µM NADPH, 1 µM FAD, 1 µM FMN, 500 µM nitro blue tetrazolium, and imidazole (0-5 mM) in a final volume of 200 µL. Product formation was monitored at 550 nm ( ) 28.6 mM-1 cm-1) using a microtiter plate reader (Dynatech MR5000) at room temperature. Enough enzyme (0.8 µg) was added

Probing the Active Site of Human iNOS

FIGURE 1: Inhibition of rH-iNOS by imidazole. Imidazole inhibited rH-iNOS in a concentration-dependent manner with a calculated IC50 of 59 ( 7 µM. Representative progress curves (inset) without added imidazole (b) and with 200 µM imidazole (3) showed no time dependence on the inhibition; thus imidazole is a reversible inhibitor of rH-iNOS.

to give a linear rate of approximately 0.005-0.010 absorbance units/min in the control sample which did not contain imidazole. Optical Spectroscopy. All spectra were recorded using a HP8425A diode-array UV/visible spectrophotometer (Hewlett Packard) equipped with a thermostated, multicell-transport cell holder connected to a RM6 Lauda refrigerated water bath. The sample spectrum was blanked against 20 mM TES, pH 7.5, 4 µM BH4 (unless otherwise indicated), and the absolute absorbance between 190 and 800 nm was recorded at 15 °C. Titration of compounds with the enzyme was accomplished by the step-wise addition of the appropriate stock solution via a syringe at 15 °C and the absolute spectrum recorded at each concentration and normalized at 700 nm. The final added volume was no more than 3% of the initial sample volume. The difference spectrum was generated by the subtraction of the resultant spectrum at each concentration of substrate or inhibitor from the initial spectrum. Binding isotherms were generated graphically by plotting the changes in absorbance at the indicated wavelengths versus the concentration of substrate or inhibitor using Sigma Plot. The dissociation constant (Kd) was calculated by fitting the data to y ) (ax)/(b + x), where y is the change in absorbance, x is the concentration of added substrate or inhibitor, a is maximum change in absorbance at infinite concentration of substrate or inhibitor, and b is the dissociation constant, Kd. RESULTS Kinetic Studies. Imidazole inhibited rH-iNOS in a concentration-dependent manner and exhibited an IC50 value of 59 ( 7 µM (Figure 1). The inhibition was not timedependent, consistent with it being a reversible inhibitor (Figure 1, inset). The increase in imidazole concentration decreased V/Karginine but not Varginine, indicating that imidazole is a competitive inhibitor of L-arginine (Figure 2). The data gave the best fit to a competitive model with a calculated

Biochemistry, Vol. 35, No. 29, 1996 9569

FIGURE 2: Effects of imidazole on the steady-state kinetic parameters of L-arginine. Imidazole at 0 µM (O), 60 µM (b), and 200 µM (3) inhibited rH-iNOS in a competitive manner with respect to L-arginine. The fit of the data to a competitive model (solid lines) yielded a Ki of 95 ( 10 µM.

Ki of 95 ( 10 µM. NOS is a self-sufficient monooxygenase containing a NADP+ reductase domain and a heme hydroxylation domain. The reductase domain has the ability to reduce certain dyes that are independent of the heme domain (Schmidt et al., 1992; Klatt et al., 1992). To test whether imidazole inhibits the reductase domain, the intrinsic diaphorase activity of the enzyme was measured as a function of imidazole concentration. The diaphorase activity was not affected at concentrations up to 1 mM, indicating that imidazole does not affect the reductase domain and suggests the site of imidazole inhibition is the heme domain (data not shown). Structure-ActiVity Relationship. A series of imidazole derivatives were tested to identify the structural requirements for inhibition of rH-iNOS. Table 1 showed that the 2- and the 4-positions of imidazole cannot be substituted without a severe loss of inhibition. However, modifications at the 1-position with alkyl and aromatic substituents were well tolerated. One of these compounds, 1-phenylimidazole inhibited rH-iNOS in a competitive manner versus L-arginine and exhibited a Ki of 38 µM (data not shown). Substitutent effects of 1-phenylimidazole were also examined. Hydroxyl and amino groups at the para position of the phenyl moiety did not change the inhibition significantly with the paraaminophenyl moiety exhibiting slightly better inhibition. However, an electron-withdrawing nitro group had a dramatic effect on the inhibition, increasing the IC50 value by 10fold. Comparison of the IC50 of 1-(2-aminoethyl)imidazole (IC50 ) 22 µM) and 2-(1-imidazol)ethanesulfonic acid (IC50 ) 4395 µM) indicates that the enzyme disfavors negatively charged substituents at N-1 position. The Effect of pH on Imidazole Inhibition. The inhibition of rH-iNOS by imidazole and 1-phenylimidazole was examined as a function of pH. Using Bis-Tris propane as a broad pH range buffer, the Ki values for imidazole and 1-phenylimidazole were measured between pH 6.5 and 9.0. Figure 3 shows that, as the pH decreased, the Ki value for imidazole increased. A fit of the data to a titration equation yielded a pKa value of 7.1 ( 0.2. In the case of 1-phen-

9570 Biochemistry, Vol. 35, No. 29, 1996

Chabin et al.

Table 1: Inhibition of rH-iNOS by Substituted Imidazole

FIGURE 3: pH dependence of the inhibition of rH-iNOS with imidazole and 1-phenylimidazole. The Ki values for imidazole (O) and 1-phenylimidazole (b) were measured as a function of pH. Imidazole showed a marked increase in the Ki value as the pH was decreased. A fit of the data to a titration equation gave a pKa value of 7.1 ( 0.2. In contrast, the Ki value of 1-phenylimidazole did not change in the same pH range examined (6.5-9.0).

ylimidazole, however, the Ki was not sensitive to the changes in the pH range examined. Comparison of the pH dependence of the Ki for imidazole to the pH independence of the Ki for 1-phenylimidazole suggests that enzymic groups have minimal contribution on the observed pKa. The Effect of BH4 on Imidazole and 1-Phenylimidazole Inhibition. The effects of exogenous BH4 concentration on the inhibition of rH-iNOS by imidazole and 1-phenylimidazole were examined. With 1-phenylimidazole, the IC50 value at 1 µM L-arginine increased from 12 ( 1 µM with no BH4 added to 84 ( 8 µM with 100 µM of added BH4. For imidazole, the IC50 value at 1 µM L-arginine increased from 12 ( 1 µM with no BH4 to 143 ( 9 µM with 100 µM of added BH4. This suggests that BH4 can affect the binding of imidazole and 1-phenylimidazole to the enzyme.3 The effects of BH4 on the inhibition of imidazole and 1-phenylimidazole were further examined with rH-iNOS expressed in a mouse NIH 3T3 cell line that is deficient in the biosynthesis of BH4 (Tzeng et al., 1995). Exogenous BH4 must be added to reconstitute enzymatic activity. This enzyme deficient in BH4 is useful in that both inhibitor and BH4 can be added at the same time in the assay and the rate would reflect the ability of the inhibitor to prevent the binding of BH4. Saturating levels of L-arginine were used in order to minimize the inhibition of L-arginine binding. Using the 3 Using immunopurified rH-iNOS, enzyme inhibition was incomplete in the absence of exogenous BH4, with 20% of the enzymatic activity not inhibited. Thus a proper fit of the IC50 equation was problematic at low BH4 concentration. Furthermore, the progress curves at low BH4 concentrations were not linear, making accurate rate determination difficult. The problem can be overcome by using rH-iNOS expressed in a NIH 3T3 cell line that is deficient in BH4 synthesis (Tzeng et al., 1995).

3T3 cytosolic fraction expressing rH-iNOS in the absence of added BH4, the rates of citrulline formation were monitored with varying imidazole or 1-phenylimidazole concentrations. Figure 4 shows that, at 100 nM BH4 and 25 µM L-arginine in the assay, both imidazole and 1-phenylimidazole inhibited citrulline formation in a concentrationdependent manner (Figure 4A,B). However, at 5 µM BH4, the inhibition was significantly attenuated for both inhibitors. The inhibition of the enzyme by N-nitro-L-arginine was not attenuated as would be expected for an inhibitor of just the L-arginine binding site (Figure 4C). This implies that imidazole and 1-phenylimidazole inhibition of rH-iNOS can be modulated by the presence of exogenous BH4. The type of inhibition exhibited by imidazole and 1-phenylimidazole was addressed with BH4 deficient rH-iNOS. The activation of the enzyme by increasing the BH4 concentration follows a typical titration curve having a Kact of 98 ( 32 nM (Figure 5). The presence of 250 µM imidazole or 50 µM 1-phenylimidazole increased the Kact by 4- and 8-fold, respectively, but did not significantly affect the Vmax. This behavior is suggestive of a competitive binding of imidazole and 1-phenylimidazole with respect to BH4. Isoform SelectiVity of Imidazole and 1-Phenylimidazole. The inhibition of the constitutive isoforms of NOS were examined for imidazole and 1-phenylimidazole. Table 2 shows imidazole at 10 µM BH4 and 1 µM L-arginine inhibited the constitutive isoforms with IC50 values of 189 ( 22 µM and 175 ( 33 µM for the rH-ecNOS and rH-ncNOS, respectively. For 1-phenylimidazole, the IC50 values were 611 ( 63 µM and 429 ( 42 µM for the rHecNOS and rH-ncNOS, respectively.

Probing the Active Site of Human iNOS

Biochemistry, Vol. 35, No. 29, 1996 9571

FIGURE 4: Effects of BH4 on the inhibition of rH-iNOS by imidazole (A), 1-phenylimidazole (B), and N-nitro-L-arginine (C). Cell lysate containing rH-iNOS expressed in murine 3T3 cell line deficient in BH4 biosynthesis was used to examine the effect of BH4 on the inhibition by these inhibitors. At 25 µM L-arginine concentration and 100 nM BH4 (O), imidazole, 1-phenylimidazole, and N-nitro-L-arginine inhibited the enzyme in a concentration-dependent manner. However, at 5 µM BH4 (b), the inhibition of the enzyme is significantly attentuated for imidazole and 1-phenylimidazole, but not for N-nitro-L-arginine.

FIGURE 5: Effects of BH4 activation of rH-iNOS by imidazole and 1-phenylimidazole. The activation of rH-iNOS expressed in 3T3 cells by increasing concentration of BH4 followed saturation kinetics exhibiting a Kact for BH4 of 98 ( 32 nM (O). Imidazole (b) and 1-phenylimidazole (3) increased the Kact for BH4 but do not significantly affect the Vmax. The data gave the best fit for a competitive model (solid lines). Table 2: Inhibition of Human NOS Isoforms by 1-Phenylimidazole and Imidazolea human isoform

IC50 (µM) 1-phenylimidazole

IC50 (µM) imidazole

rH-iNOS rH-ecNOS rH-ncNOS

33 ( 3 611 ( 63 429 ( 42

59 ( 7 189 ( 22 175 ( 33

a rH-iNOS is immunoaffinity/DEAE purified; the cNOSs were from spin-filtered lysates. BH4 concentration was 10 µM in all cases.

Spectroscopic Studies. rH-iNOS exhibited a typical Soret absorbance having a maximum at 400 nm with other absorbance features at 450 nm due to the flavins.2 The sequential addition of increasing concentrations of imidazole to rH-iNOS caused an incremental red shift of the Soret maximum from 400 to 430 nm. This concentration-dependent absorbance change is typical of type II difference spectra

FIGURE 6: Spectral perturbation of the Soret upon binding of imidazole to rH-iNOS. Imidazole elicited a concentration dependent optical absorbance change of the enzyme bound heme. The difference spectra were characteristic of a type II spectral change that had a peak at 422 nm and a trough at 390 nm. A plot of the magnitude of the absorbance change at 422 nm and 390 nm as a function of imidazole concentration yielded a saturation curve with a calculated Kd for imidazole of 63 ( 2 µM (inset).

and is characterized by a peak at 430 nm and a trough at 390 nm (Figure 6). This indicates that imidazole binding causes the population of high spin heme iron to convert to a low spin state upon binding of imidazole (McMillan & Masters, 1993). Measurements of the magnitude of the type II spectral change as a function of imidazole concentration allowed for the determination of the Kd for imidazole to be 63 ( 2 µM (Figure 6, inset). This is in good agreement with the Ki for imidazole of 95 µM determined from steadystate kinetics. Spectral titration of the free enzyme with L-arginine yielded a type I difference spectrum with a peak at 390 nm and a trough at 422 nm, similar to the rat ncNOS (McMillan & Masters, 1993). The calculated Kd value for L-arginine is 3.0 ( 0.4 µM, similar to the steady-state Km of 2.3 µM (Figure 7A). The addition of increasing concen-

9572 Biochemistry, Vol. 35, No. 29, 1996

Chabin et al.

FIGURE 7: Spectral perturbation of the Soret upon binding of L-arginine to rH-iNOS. (A) L-Arginine caused a concentration-dependent optical absorbance change of the enzyme-bound heme. The difference spectra had characteristics of a type I spectral change with a peak at 390 nm and a trough at 422 nm. A plot of the magnitude of the absorbance change at 390 and 422 nm as a function of L-arginine concentration yielded a titration curve with a calculated Kd of 3.03 ( 0.45 µM (inset). (B) L-arginine also caused a type I spectral change of the enzyme-bound heme in the presence of 2.2 mM imidazole, but the apparent Kd for L-arginine under these conditions was 209 ( 10 µM (inset).

FIGURE 8: Spectral perturbation of the Soret upon binding of 1-phenylimidazole to rH-iNOS. (A) 1-phenylimidazole caused a type II spectral change with a calculated Kd of 28 ( 3 µM (inset). (B) In the presence of 1.6 mM 1-phenylimidazole, L-arginine caused a type I difference spectra with an apparent Kd of 46 ( 7 µM.

tration of L-arginine to the imidazole-enzyme complex also resulted in a type I spectral change (Figure 7B). However, the measurement of the magnitude of the type I spectral change as a function of L-arginine concentration gave a Kd value for L-arginine of 209 ( 10 µM (Figure 7B, inset). Thus the Kd for L-arginine was 70-fold higher in the presence of 2.2 mM imidazole compared to the value in the absence of imidazole. The addition of 1-phenylimidazole to rH-iNOS also caused a type II difference spectrum (Figure 8A). The Kd for 1-phenylimidazole was 28 ( 3 µM at 4 µM BH4 (Figure 8A, inset). This compares well with the steady-state Ki of 38 ( 4 µM. The titration of the 1-phenylimidazole-enzyme complex with L-arginine gave a type I difference spectrum. The apparent Kd for L-arginine is 46 ( 7 µM in the presence of 1.6 mM 1-phenylimidazole (Figure 8B). This value was ∼15-fold higher than the value of 3 µM measured in the absence of inhibitor (Figure 7A). The perturbation of the

Kd value of L-arginine by imidazole and 1-phenylimidazole is in accord with the competitive nature of these inhibitors observed from steady-state kinetics. DISCUSSION Imidazole is a classical inhibitor of cytochrome P450 enzymes. It exerts its inhibitory effect via the direct ligation to the heme iron at the dioxygen binding site (Lipscomb, 1980; Shiro et al., 1989; Banci et al., 1994). The rH-iNOS has been shown to possess a cytochrome P450-like heme in the active site as does the murine iNOS counterpart (White & Marletta, 1992). Studies reported here demonstrate imidazole is a competitive inhibitor of L-arginine having a Ki of 95 µM. Spectrophotometric binding studies demonstrate that imidazole binds to the enzyme causing a type II optical difference spectrum with a Kd of 63 µM, indicative of its interaction with the heme iron at the sixth axial ligand site (McMillan & Masters, 1993). Saturating concentration

Probing the Active Site of Human iNOS of imidazole (2.2 mM) also perturbs the binding affinity of L-arginine, increasing the Kd by about 70-fold, from 3 to 209 µM. These data are in accord with the steady-state kinetic results and indicate that both imidazole and L-arginine bind to a mutually exclusive site. The 2-fold more potent inhibitor, 1-phenylimidazole, is also a competitive inhibitor versus L-arginine based on the same type of analysis. The competitive behavior of imidazole with respect to L-arginine suggests a link between the binding site of imidazole (dioxygen site) and L-arginine, but the two compounds perturb the active site heme differently. The binding of L-arginine to the enzyme induces a blue-shift in the Soret of the imidazole-enzyme complex from 422 to 396 nm, indicating a change in the coordination state of the heme ferric iron upon binding. The 430 nm species is a six-coordinate low spin heme with imidazole as the sixth axial ligand, and the 396 nm species is the five-coordinated high spin heme (McMillan & Masters, 1993). Upon binding of L-arginine to the imidazole-rH-iNOS binary complex, the ligation of imidazole to the ferric iron is disrupted as evident from the spectral shift. The observed competitive nature of imidazole with respect to L-arginine suggests that imidazole is subsequently displaced from the active site upon L-arginine binding. This change in coordination state of the heme as a result of substrate binding is similar to the binding of the substrate, camphor, to the low spin six-coordinate heme of P450cam (Poulos et al., 1985, 1986). In this case a water molecule is the sixth axial ligand and is displaced upon substrate binding. Imidazole has been reported to inhibit NOS from different species (Wolff et al., 1993; Wolff & Gribin, 1994a,b; Mayer et al., 1994). The rat neuronal cNOS has been extensively characterized by kinetic and spectrophotometric methods (Wolff et al., 1993; McMillan & Masters, 1993). For this enzyme, imidazole is a noncompetitive inhibitor versus L-arginine exhibiting a Ki of 200 µM. A noncompetitive pattern with a Ki of 48 µM has also been observed for the murine macrophage iNOS based on steady-state kinetic studies (Wolff & Gribin, 1994a). The noncompetitive patterns for these two enzymes suggest that imidazole is not displaced from the active site by the binding of L-arginine, although the optical spectroscopic data suggest a disruption of the sixth axial ligand (imidazole) leading to a fivecoordinate high spin heme (McMillan & Masters, 1993). Thus a ternary complex between the enzyme, imidazole, and L-arginine exists. On the other hand, competitive patterns versus L-arginine have been observed for the bovine endothelial and porcine neuronal cNOSs with Ki values of 50 and 198 µM, respectively (Wolff & Gribin, 1994a; Wolff et al., 1994; Mayer et al., 1994). These observed variations in the kinetic parameters as well as in the type of inhibition exerted by imidazole strongly suggest that there are microheterogeneities at the heme hydroxylation site among these enzymes from different species. Furthermore, in the case of the rat brain cNOS and murine iNOS, there may exist interactions that prevent the displacement of imidazole from the active site, even though the ligation to the heme is disrupted upon binding of L-arginine. These interactions are absent in rH-iNOS as well as in the porcine neuronal and bovine endothelial cNOSs. These observed differences in the inhibition profile of imidazole derivatives between rHiNOS and murine macrophage iNOS are noteworthy and have been observed for another inhibitor, aminoguanidine

Biochemistry, Vol. 35, No. 29, 1996 9573 (Wolff & Lubbeskie, 1995; Garvey et al., 1994). Thus extrapolations of inhibition data derived from the murine macrophage iNOS to the human iNOS should be made with caution. Imidazole is the only inhibitor of NOS in which the mode of binding has been firmly established, based on the wealth of kinetic and spectroscopic data. Thus, it is an attractive candidate for probing the active site heme domain of rHiNOS. A combination of structure-activity relationship (SAR) studies and pH profiles of inhibition with imidazole and its derivatives suggest the structural and electronic requirements for optimal inhibition. The pH dependence of the inhibition of the enzyme indicates an ionizable group is deprotonated for maximal inhibition. However, in the case of 1-phenylimidazole, the Ki value was 2-fold lower than imidazole and exhibited no pH dependence. Differences in the mode of binding cannot account for this observation, since spectroscopic studies indicate the binding of 1-phenylimidazole to the enzyme is qualitatively indistinguishable from that of imidazole. Thus, one can rule out significant involvement of enzymic groups to the observed pKa. The identity of this ionizable group is unknown at this point; however, an assignment of the observed pKa to that of imidazole seems reasonable. This would also explain why enzyme inhibition with 1-phenylimidazole is pH independent since its pKa is 5.8 (Elguero et al., 1968), 1.1 pH unit lower than imidazole (pKa ) 6.9). Thus at pH 6.5, the neutral form of 1-phenylimidazole predominates over the protonated form by 5 to 1; in contrast to imidazole where, at the same pH, the protonated form predominates over the unprotonated form by 2.5 to 1. If this is correct, then this pKa effect emphasizes the importance of the protonation state of the N-3 nitrogen of imidazole in the inhibition of rH-iNOS. Consistent with this hypothesis is the observation that 1-(pnitro)phenylimidazole is a weak inhibitor. This may be probably due to the electron withdrawing ability of the nitro group that decreases the electron density at N-3 resulting in the reduced ability to bind to the heme. Finally, alkyl substituents at the 2- or the 4-position probably interfere with iron binding due to steric reasons. Similar SAR results have been observed with imidazole and its derivatives in the inhibition of microsomal cytochrome P450 enzymes and provide additional evidence that the heme of rH-iNOS is cytochrome P450-like (Rogerson et al., 1977). The requirement for BH4 for stability and maximal activity is a unique property of NOS and sets it apart from all P450 enzymes. Although the relationship between BH4 and protein dimer integrity has been established for murine and human iNOS (Baek et al., 1993; Tzeng et al., 1995), the mechanistic link between this cofactor and NO formation relative to the P450 heme is unclear. For rH-iNOS, imidazole and 1-phenylimidazole inhibition can be affected by exogenous BH4. Furthermore, both imidazole and 1-phenylimidazole behave as competitive inhibitors with respect to enzyme activation by exogenous BH4. This competitive behavior of imidazole with both L-arginine and BH4 has not been observed previously with any inducible NOS isoforms and suggests the BH4 binding site is very close to the dioxygen site (Table 3). Exogenous BH4 had no effect on the inhibition of the enzyme by N-nitro-L-arginine, indicating that the guanidino moiety does not overlap with the BH4 site. A speculative model of the heme active site of rHiNOS is shown in Figure 9. The close convergence of both

9574 Biochemistry, Vol. 35, No. 29, 1996

Chabin et al.

Table 3: Inhibition Parameters of Imidazoles for NOS Isoforms from Different Species inhibition pattern inhibitor imidazole

1-phenylimidazole

d

NOS isoform bovine ncNOS murine iNOS bovine ecNOS human iNOS bovine ncNOS murine iNOS bovine ecNOS human iNOS

L-Arg a

NC NC Cb C NC NC C C

BH4

Ki (µM)

NC NC NC C NC C C C

200c 48d 50e 95f 38c 0.7d 50e 38f

a NC, noncompetitive. b C, competitive. c From Wolff et al. (1993). From Wolff et al. (1994a). e From Wolff et al. (1994b). f This report.

selectivity. The use of a photoaffinity probe such as 1-(4azidophenyl)-imidazole may be useful in the identification of amino acid residues that interact with the phenyl ring. These experiments are in progress. In summary, we have described the detailed kinetic and spectroscopic studies of human iNOS with respect to imidazole inhibition. These studies have provided initial insights into the relative spatial arrangements of the substrate binding sites relative to the P450-like heme of this enzyme. The wide spectrum of inhibition patterns observed for the imidazole class of inhibitors reflects the differences in the relative spatial arrangements of the L-arginine, dioxygen, and BH4 binding sites among NOS isoforms from different species. ACKNOWLEDGMENT We thank Dr. T. Billiar of the University of Pittsburg for the cell lysates of murine 3T3 cell line expressing rH-iNOS and Drs. Philip Davies, Michael Tocci, and Mr. Richard Mumford for their support. REFERENCES

FIGURE 9: Proposed arrangement of the three binding sites in rHiNOS.

the BH4 and L-arginine sites into the dioxygen site of the heme is the notable feature and is consistent with the data presented here. We suggest that the variations in the inhibition parameters of imidazole observed with different NOS’s are due to the difference in the spatial arrangement of the BH4 and L-arginine sites relative to the P450 heme. The close proximity of the L-arginine binding site to the heme site makes mechanistic sense since hydroxylation would require the heme. However, the close proximity of the BH4 site to the heme brings into question the catalytic role of BH4 in the hydroxylation of L-arginine. Additional studies are needed to answer this question. The intimate arrangement of the three substrate sites in rH-iNOS may have implications in the design of multisubstrate site inhibitors. An important issue in the development of an inhibitor for iNOS is isoform selectivity. 1-Phenylimidazole is 20-fold selective for rH-iNOS over rH-ecNOS. This selectivity, although modest, suggests isoform variations between the active sites of the human iNOS and ecNOS. It is tempting to speculate the existence of a specific interaction(s) between rH-iNOS and the phenyl ring that gives rise to the observed

Baek, K., J., Theil, B. A., Lucas, S., & Stuehr, D. J. (1993) J. Biol. Chem. 268, 21120-21129. Banci, L., Bertini, I., Marconi, S., Pierattelli, R., & Sligar, S. G. (1994) J. Am. Chem. Soc. 116, 4866-4873. Bredt, D. S., & Synder, S. H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 682-685. Cash, J. M., & Klippel, J. H. (1994) New Eng. J. Med. 330, 13681375. Cleland, W. W. (1979) Methods Enzymol. 63, 104-120. Cho, H. J., Xie, Q.-w., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., & Nathan, C. F. (1992) J. Exp. Med. 176, 599-604. Elguero, J., Gonzolez, E., Imbach, J. L., & Jacquier, R. (1968) Bull. Soc. Chim. Fr. 12, 5017-5019. Garvey, E. P., Oplinger, J. A., Tanoury, G. J., Sherman, P. A., Fowle, M., Marshall, S., Harmon, M. F., Paith, J. E., & Furfine, E. S. (1994) J. Biol. Chem. 43, 26669-26676. Granger, D. L., & Lehninger, A. L. (1982) J. Cell. Biol. 95, 527535. Hevel, J. M., & Marletta, M., A. (1992) Biochemistry 31, 71607165. Klatt, P., Heinzel, B., John, M., Kastner, M., Bohme, E., & Mayer, B. (1992) J. Biol. Chem. 267, 11374-11378. Lepoivre, M., Flaman, J.-M., & Henry, Y. (1992) J. Biol. Chem. 267, 22994-23000. Lipscomb, J. D. (1980) Biochemistry 19, 3590-3599. Mayer, B., & Werner, E. R. (1995) Naunyn-Schmiedeberg’s Arch. Pharmacol. 351, 453-463. Mayer, B., Klatt, P., Werner, E. R., & Schmidt, K. (1994) FEBS Lett. 350, 199-202. McMillan, K., & Masters, B. S. S. (1993) Biochemistry 32, 98759880. Moncada, S., & Higgs, A. (1993) New Eng. J. Med. 329, 20022012. Mulligan, M. S., Heirel, J. M., Marletta., M. A., & Ward, P. A. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6331-6342. Nakaki, T., Nakayama, M., & Kato, R. (1990) Eur. J. Pharmacol. 189, 347-353. Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., & Kraut, J. (1985) J. Biol. Chem. 260, 16122-16128. Poulos, T. L., Finzel, B. C., & Howard, A. J. (1986) Biochemistry 25, 5314-5319. Robertson, C. A., Green, B. G., Niedzwiecki, L., Harrison, R. K., & Grant, S. K. (1993) Biochem. Biophys. Res. Commun. 197, 523-528. Rogerson, T. D., Wilkinson, C. F., & Hetarski, K. (1977) Biochem. Pharmacol. 26, 1039-1042. Schmidt, H. H. H., Smith, R. M., Nakane, M., & Murad, F. (1992) Biochemistry 31, 3243-3249.

Probing the Active Site of Human iNOS Segal, I. H. (1975) in Enzyme Kinetics, BehaVior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, pp 100125, John Wiley & Sons, New York. Shiro, Y., Iizuka, T., Makino, R., Ishumura, Y., & Morishima, I. (1989) J. Am. Chem. Soc. 111, 7707-7710. Stuehr, D. J., & Ikeda-Saito, M. (1992) J. Biol. Chem. 267, 2054720550. Tzeng, E., Billiar, T. R., Robbins., P. D., Loftus, M., & Stuher, D. J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11771-11775. White, K. A., & Marletta, M. A. (1992) Biochemistry 31, 66276631.

Biochemistry, Vol. 35, No. 29, 1996 9575 Wolff, D. J., & Gribin, B. J. (1994a) Arch. Biochem. Biophys. 311, 293-299. Wolff, D. J., & Gribin, B. J. (1994b) Arch. Biochem. Biophys. 311, 200-306. Wolff, W. J., & Lubbeskie, A. (1995) Arch. Biochem. Biophys. 316, 290-301. Wolff, D. J., Datto, G. A., Samatovicz, R. A., & Tempsick, R. A. (1993) J. Biol. Chem. 268, 9825-9429. Wolff, W. J., Lubbeskie, A., & Umansky, S. (1994) Arch. Biochem. Biophys. 314, 360-366. BI960476O